Hidden on the frontiers: Calotes vultuosus (Harlan, 1825) (Squamata, Agamidae), a species new for the reptile list of Pakistan

the journal of biodiversity data NOTES ON GEOGRAPHIC DISTRIBUTION 1207 Academic editor: Abhijeet Bayani Received: 20 September 2025 Accepted: 13 November 2025 Published: 8 December 2025 Copyright © The authors. This is an open‑access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0) Abstract. We document the first confirmed record of Calotes vultuosus (Harlan, 1825) from Pakistan, based on three specimens from Keamari, Sindh. DNA barcodes (16S, COI) and morphological traits place the studied material firmly within C. vultuosus, clearly distinct from related congeners. This finding extends the species’ distribution westward from India into coastal Pakistan and highlights the biogeographic significance of southern Pakistan and integrative approaches for resolving the distribution of the C. versicolor complex in South Asia. Key words. DNA barcoding, first record, Sindh, South Asia, taxonomy Jablonski D, Kousar M, Masroor R (2025) Hidden on the frontiers: Calotes vultuosus (Harlan, 1825) (Squamata, Agamidae), a species new for the reptile list of Pakistan. Check List 21 (6): 1207–1212. https://doi.org/10.15560/21.6.1207 Introduction The reptile diversity of South Asia, particularly Pakistan, is high but remains greatly underestimated (Khan 2006; Masroor 2012). The country spans a vast territory encompassing habitats from extreme deserts to high-elevation mountains and lies at the intersection of two (Khan 2006) or three (Holt et al. 2013) major terrestrial zoogeographical realms: the Palearctic, Saharo-Arabian, and Oriental realms. This unique position has allowed faunal elements from these realms to meet, interact, and evolve, making the region a natural laboratory for studying ongoing speciation and colonization processes. However, given the limited resources available for biodiversity research, reliable estimates of reptile diversity remain challenging. In recent years, DNA data from local amphibian and reptile populations have substantially improved our understanding of vertebrate diversity between the Hindu Kush, Himalaya, and the Arabian Sea (e.g. Jablonski et al. 2020, 2025; Masroor et al. 2022; Amarasinghe et al. 2023; Hofmann et al. 2023). These advances also highlight several reptile groups in need of further taxonomic and biogeographic attention, including the Agamidae, one of the most diverse families in the region. Pakistan hosts multiple agamid genera that display notable ecological and phenotypic variation. Among these genera, for example, Laudakia Gray, 1845 comprises large-bodied lizards with high species diversity across both montane habitats and the arid landscapes, yet their genetic structure and distribution patterns remain insufficiently explored. A similarly incomplete picture is evident in Calotes Cuvier, 1817, which reaches its westernmost distribution along the eastern foothills of the Hindu Kush and the Iranian Plateau, but whose regional diversity and phylogeographic history are still poorly known. Traditionally, most populations of Calotes in the Indian subcontinent were identified as C. versicolor (Daudin, 1802). However, an integrative study combining morphological and genetic data (Gowande et al. 2021) demonstrated that this taxon represents a complex of four distinct species distributed across South Asia. Of these, two occur within Pakistan or in its proximity in India. The first, C. farooqi Auffenberg & Rehman, 1993, inhabits hilly areas of northern Pakistan and eastern Afghanistan (and was surprisingly detected in one Sindh population), while C. vultuosus (Harlan, 1825) is distributed widely in central and northern India, including Gujarat and Rajasthan, near the Pakistani border. Conversely, C. versicolor sensu stricto, which is restricted to southern and eastern India and introduced to 21 (6) · https://doi.org/10.15560/21.6.1207 21 (6): 1207–1212. https://doi.org/10.15560/21.6.1207 Hidden on the frontiers: Calotes vultuosus (Harlan, 1825) (Squamata, Agamidae), a species new for the reptile list of Pakistan Daniel Jablonski1, Misbah Kousar1, Rafaqat Masroor2 1 Department of Zoology, Comenius University in Bratislava, Bratislava, Slovakia 2 Zoological Sciences Division, Pakistani Museum of Natural History (PMNH), Islamabad, Pakistan Corresponding author: Daniel Jablonski ([email protected]) Check List 21 (6) · https://doi.org/10.15560/21.6.1207 Jablonski et al. · Calotes vultuosus in Pakistan 1208 the Maldives, does not occur in Pakistan (Gowande et al. 2021). Despite these advances, the distribution and diversity of Calotes in Pakistan remain poorly understood, and it is unclear whether one or more species are present. To address this question, we collected and analysed specimens from Sindh Province using both morphological and genetic data. Methods During fieldwork in the coastal areas of Sindh and Balochistan, Pakistan, in 2021, we collected three specimens of the genus Calotes near Keamari village (Karachi Division) for further examination (Figure 1A). Geographic coordinates were recorded using the WGS84 datum. The collection comprised two adults and one subadult. Specimens were euthanized and preserved in 70% ethanol for long-term storage in the personal collection of the first author (Comenius University Herpetological Collection, CUHC), with tissue samples for DNA analysis preserved separately in 96% ethanol. In addition, we recorded 37 morphological characters (Table 1), as defined by Zug et al. (2007), from newly available specimens included in this study. To visualize the distribution of the species and update its known range, we prepared a distribution map using QGIS v. 3.44 Solothurn (QGIS Development Team 2025) and morphologically and genetically verified records from Gowande et al. (2021). Based on previous research and the availability of comparative datasets, we generated new mitochondrial sequences of 16S rRNA (16S) and cytochrome oxidase subunit I (COI) using primers and PCR conditions described by Gowande et al. (2021). Total genomic DNA was extracted with the E.Z.N.A.® Plasmid DNA Maxi Kit, following the manufacturer’s protocol. Sequences were obtained both from the newly collected material from Keamari village and from two specimens of C. farooqi from northern Pakistan (CUHC 10228, 10247), which had previously been sequenced only for 16S (Gowande et al. 2021). For accurate species assignment based on mitochondrial sequences, we included material originating from or near the type localities of related South Asian congeners (Figure 1A), as reported by Gowande et al. (2021). The comparative dataset comprised DNA material of C. farooqi (CUHC 10228, 10247), C. irawadi (AG855, CAS 204862), C. versicolor (NCBS AT102), and C. vultuosus (CESL 1063, 1081). The following sequences were used for comparison: MW901313–14 (C. farooqi), MW901300–01 (C. Figure 1. Distribution and first confirmation of Calotes vultuosus for Pakistan. A. Map showing the new locality record from Keamari, Karachi Division, Sindh Province, Pakistan (blue circle), with additional comparative localities and type locality of the species (data from Gowande et al. 2021). Question marks indicate areas with uncertain species affiliation. B. Adult female Calotes vultuosus (CUHC 11172) from Keamari, Karachi Division, Sindh Province, Pakistan. C.Phylogenetic network based on concatenated mitochondrial 16S rRNA (524 bp) and COI (608 bp) sequences, generated in SplitsTree, showing the placement of Pakistani specimens (CUHC 11167, CUHC 11168, CUHC 11172) within C. vultuosus (blue), distinct from C. farooqi (green), C. irawadi (yellow), and C. versicolor (red). Scale bar indicates genetic distance. Check List 21 (6) · https://doi.org/10.15560/21.6.1207 Jablonski et al. · Calotes vultuosus in Pakistan 1209 irawadi), MW901307 (C. versicolor), and MW901298–99 (C. vultuosus) for 16S; MZ489212 (C. irawadi), MZ489209 (C. versicolor), and MZ489206 (C. vultuosus) for COI. Two COI sequences of C. farooqi (CUHC 10228, 10247) were newly produced in this study. As an outgroup, we used 16S and COI genes extracted from the mitogenome sequence of Laudakia tuberculata (OR887527). Newly generated sequences have been deposited in GenBank under the accession numbers PX418822–24 (16S) and PX418913–17 (COI). Sequences of 16S (524 bp) and COI (608 bp) were trimmed in Seaview (Gouy et al. 2021), concatenated, and analyzed using a phylogenetic network constructed in SplitsTree v. 4 (Huson and Bryant 2006) based on uncorrected p-distances. Results Order Squamata Family Agamidae Genus Calotes Cuvier, 1817 Calotes vultuosus (Harlan, 1825) New records. PAKISTAN — Sindh Province • Karachi Division, Keamari District, Keamari; 24.8842°N, 66.8159°E; alt. 29 m; 14.IX.2021; Jablonski D, Masroor R. leg.; semi-arid lowland with sandy–loamy soil, scattered Acacia trees, grass patches, and an open cultivated–natural ecotone near low rocky hills; 2 adult ♀ CUHC 11167, 11172 (Figure 1B), GenBank: PX418822, PX418824 (16S), PX418915, PX418917 (COI), 1 sex indet., subadult, CUHC 11168, GenBank: PX418823 (16S), PX418916 (COI). Table 1. Morphological data for 37 characters collected from Calotes vultuosus from Keamari, Sindh Province, Pakistan. Measurements in mm. Character Voucher CUHC 11167 CUHC 11168 CUHC 11172 Sex Female ?Female Eye–ear length 5.8 4.2 7.3 Head height 19.9 17.5 21.8 Head length 26.3 23.0 35.3 Head width 22.4 14.6 26.0 Interorbital width 11.8 9.8 13.3 Jaw width 16.3 13.2 22.6 Naris–eye length 3.8 3.4 5.0 Snout–eye length 9.0 7.1 10.9 Snout width 4.5 4.4 5.1 4thfinger 10.1 10.0 12.1 4th toe 16.4 14.7 19.6 Crus length 20.0 17.5 26.1 Forefoot length 17.9 15.4 19.9 Hindfoot length 27.3 23.7 29.3 Lower arm length 14.4 14.1 18.5 Pectoral width 15.8 13.3 21.9 Pelvic width 12.7 7.9 16.1 Snout–vent length 87.7 78.5 115.6 Snout–forelimb length 35.9 26.8 39.0 Tail height 8.4 7.0 15.3 Tail length 225 155 161 (incomplete) Tail width 11.8 7.4 14.1 Trunk length 45.9 42.5 67.5 Upper arm length 17.9 14.9 23.8 Upper leg length 21.7 19.2 24.3 Canthus rostralis 888 Dorsal eyelid scales 12 10 13 Dorsal head scales 14 13 14 Head scales 12 11 13 Infralabials 12/13 11/11 11/11 Snout scales 6 6 6 Supralabials 12/11 12/11 12/12 Temporal spines 222 4thfinger lamellae 18 17 18 4thtoe lamellae 25 24 25 Dorsal scales 43 42 46 Mid-body scale rows 42 42 43 Check List 21 (6) · https://doi.org/10.15560/21.6.1207 Jablonski et al. · Calotes vultuosus in Pakistan 1210 Identification. Molecular analyses of mitochondrial markers (concatenated 16S rRNA, COI) place the Pakistani specimens firmly within C. vultuosus, clearly distinct from C. farooqi, C. irawadi, and C. versicolor (Figure 1C). The specimens from Keamari form a well-supported clade with Indian sequences of C. vultuosus (p-distance < 1%). This represents the first confirmed record of C. vultuosus from Pakistan, extending the known distribution of the species westward from India to the coastal Sindh region. Metric and meristic characteristics obtained (Table 1) support the species identification. Morphological examination of the three specimens from Keamari (CUHC 11167, 11168, 11172) corresponds well with the diagnosis of C. vultuosus as presented by Gowande et al. (2021). Key characters such as midbody scale rows (42–43), dorsal scales (42–46), supralabials (11–12), infralabials (11–13), and two temporal spines are fully consistent with the published ranges. Subdigital lamellae on the fourth finger (17–18) and fourth toe (24–25) fall at the lower end of the reported variation (19–22 and 23–28, respectively) but still overlap with the species’ limits. Snout–vent length of the two females (87.7–115.6 mm) also matches the values known for adult C. vultuosus, with the largest specimen slightly exceeding the mean size reported for females. Taken together, both meristic and morphometric data clearly support the identification of the Pakistani specimens as C. vultuosus. Discussion Our study provides the first confirmed record of Calotes vultuosus in Pakistan, extending the known range of the species westward from India into the Sindh coastal region and likely further into Pakistan through mainland Sindh and Balochistan, where it appears to be restricted to oases and watercourse margins (Minton 1966; Mertens 1969; Sindaco and Jeremcenko 2008). The specimens from Keamari are morphologically and genetically consistent with C. vultuosus as defined by Gowande et al. (2021). Meristic traits (e.g. midbody scale rows, dorsal scales, temporal spines), mitochondrial DNA data (16S and COI), and geographical context clearly distinguish the Pakistani specimens from C. farooqi (northern Pakistan, eastern Afghanistan), C. irawadi (Myanmar and Indochina), and C. versicolor (southern India and the Maldives). The presence of C. vultuosus in southern Pakistan indicates that the species has a broader distribution than previously recognized (Gowande et al. 2021) and underscores the importance of the semi-arid plains and coastal areas of southern Pakistan as both colonization corridors and contact zones between different zoogeographic realms (Agarwal et al. 2022; Jablonski et al. 2025). The record from Keamari village highlights such biogeographic significance of this region, suggesting that similar cases of past colonization from South Asia into the Palearctic (west of the Indus River) may be revealed in other widely dispersing reptile species once genetic data are applied. In this context, a key question concerns the species identity of the regionally westernmost Calotes populations reported from Iran’s Sistan and Balochistan Province (Mobaraki et al. 2013). Based on morphological traits visible in available photographs (see also iNaturalist 2023), this part of Iran is likely inhabited by C. vultuosus rather than C. versicolor. Although this would not be unexpected from a biogeographic perspective (see distribution of Euphlyctis adolfi, Varanus bengalensis or Bungarus persicus; Kamali 2020; Safaei-Mahroo et al. 2023; Arianejad et al. 2025) genetic confirmation is still required. Our discovery also underscores the need for continued, rigorous herpetological exploration in Pakistan, where several lineages of the C. versicolor complex occur in proximity (Gowande et al. 2021). The recently elevated C. farooqi is restricted to the hilly regions of northern Pakistan and eastern Afghanistan, whereas C. vultuosus now appears to occupy the lowland coastal areas of Sindh and likely Balochistan Provinces. We also expect its presence in the lowland areas of Punjab Province, although this has not yet been genetically confirmed. Photographic records on iNaturalist (2021) and the species documented presence on the Indian side of the border (Figure 1A; Gowande et al. 2021), however, suggest that its distribution in Pakistan might be broader than currently recognized, highlighting the need for further field surveys and genetic sampling. More broadly, the distribution of Calotes from the foothills of Hindu Kush and Himalaya to the Indian plains remains unresolved, and the extent of contact zones or possible hybridization between species in this complex has yet to be studied. In this context, the genetic affiliation of the population from Mithi, Sindh (loc. 79 in Gowande et al. 2021), remains unclear. Although it unexpectedly clusters with the lineage related to C. farooqi, its geographic position suggests (cf. Figure 1A) that it should fall within the expected range of C. vultuosus. This discrepancy warrants further investigations. In conclusion, the documentation of C. vultuosus in Pakistan not only fills a distributional gap for this taxon but also emphasizes the significance of integrative taxonomy combining morphological and molecular evidence for correctly identifying cryptic members of the C. versicolor complex and Check List 21 (6) · https://doi.org/10.15560/21.6.1207 Jablonski et al. · Calotes vultuosus in Pakistan 1211 their accurate distribution in South Asia. Further surveys across Balochistan and adjoining regions are expected to clarify whether C. vultuosus is restricted or more widespread species of southwestern Pakistan. Acknowledgements We thank all colleagues who assisted us during fieldwork in southern Pakistan, Jana Poláková for her support in the DNA laboratory, and the reviewer Lukáš Pola for his valuable comments on the manuscript. Additional information Conflict of interest The authors declare that no competing interests exist. Ethical statement No ethical statement is reported. Funding Daniel Jablonski was supported by the grant of the Scientific Grant Agency of the Slovak Republic VEGA 1/0391/25 and by the EU NextGenerationEU scholarship through the Recovery and Resilience Plan for Slovakia under the project No. 09I03-03-V04-00306. Author contributions Conceptualization: DJ. Data curation: all authors. Formal analysis: DJ, MK. Funding acquisition: DJ. Investigation: all authors. Methodology: DJ. Resources: DJ. Visualization: DJ. Writing – original draft: DJ. Writing – review and editing: all authors. Author ORCID iDs Daniel Jablonski https://orcid.org/0000-0002-5394-0114 Misbah Kousar https://orcid.org/0009-0009-5342-0723 Rafaqat Masroor https://orcid.org/0000-0001-6248-546X Data availability All data that support the findings of this study are available in the main text or GenBank depository. 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